Bacteroids fragilis is an important human pathogen. Attempts to evaluate and investigate determinants that may contribute to the virulence of this organism have been hampered by the absence of suitable systems of genetic analysis. We propose to continue the development of genetic systems in this organism with two specific aims. To investigate the determinants of antibiotic resistance and their transfer within B. fragilis and from B. fragilis to E. coli. We will characterize pBFTM10, a 15 kb transfer factor that encodes Clnr in B. fragilis and Tetr in E. coli. These resistances are carried on a transposon, Tn4400, that functions in E. coli. Studies are described to determine the transposition properties of this element and the IS elements which are present as direct repeats at each end. Studies will be directed towards understanding the "tetracycline transfer factor" that promotes plasmid and transposon transfer between B. fragilis strains and into E. coli. The second aspect of our proposal is to develop a genetic system to analyze virulence factors in B. fragilis. This will involve isolating mutants of selected phenotypic characteristics and then isolating the gene(s) encoding the determinants by making use of a chromosome library of B. fragilis cloned in E. coli. Tn4400 will be employed as a mutagen in B. fragilis to help locate chromosomal genes suspected to be virulence factors. The determinants to be investigated are imipenem and tetracycline resistance, catalase production and neuraminidase production. The beta-lactamase that inactivates imipenem will be studied first. The genes encoding tetracycline resistance in B. fragilis will be studied and we will attempt to develop a probe to study the mechanism of Tetr transfer. The role of catalase in the ability of B. fragilis to survive in oxygenated tissue will be assessed. Finally, the role that neuraminidase plays in tissue destruction during B. fragilis infections will be investigated. Once the mutants have been isolated and the gene(s) encoding the determinant are located then important questions related to expression can be addressed. The results of these studies should give a clearer picture of how DNA is transferred in B. fragilis and help to provide a genetic system to study virulence factors in this important pathogen.